Figure 4: PIAS3 serves as a SUMO E3 ligase of DBC1.
From: Modification of DBC1 by SUMO2/3 is crucial for p53-mediated apoptosis in response to DNA damage
(a) HisMax-DBC1 was expressed in HEK293T cells with each of Flag-tagged PIAS1-4. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblot analysis. (b) HeLa cells incubated with and without etoposide for 36 h were subjected to immunoprecipitation with anti-DBC1 antibody followed by immunoblot analysis (left). (c) Flag-tagged SUMO3 and Ubc9 were expressed in HEK293T cells with HisMax-DBC1 and/or Myc-PIAS3. Cell lysates were subjected to pull-down with NTA-resins followed by immunoblot analysis. (d) HeLa cells expressing nonspecific shRNA (shNS) or shPIAS3 were incubated with and without etoposide for 36 h. Their lysates were then treated as in b. (e) shNS and shPIAS3 were expressed in HeLa cells with and without Myc-PIAS3. After incubation with and without etoposide for 36 h, cell lysates were subjected to immunoprecipitation with anti-SIRT1 antibody followed by immunoblot analysis. ‘i’ and ‘e’ denote shRNA insensitive and endogenous, respectively (left).
